Assembly and Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator and Associated Proteins

Cystic Fibrosis (CF) is an autosomal recessive genetic disease that leads to severe malfunction in many organs, but particularly the lungs. The primary cause of this malfunction is the decrease of the airway surface liquid layer on the lung epithelium. The lack of hydration leads to mucus build up on the epithelial lining, leading to blockage of airways. The underlying cause of CF is the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), which results from mutations in the protein. Almost 90% of CF patients are caused by the deletion of the phenylalanine at position 508 of CFTR, which is believed to affect the folding and stability of CFTR. The misfolded ΔF508-CFTR undergoes ER associated degradation (ERAD), causing the failure of ΔF508-CFTR trafficking to the cell surface. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. In this dissertation, real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however the amount of channel in each vesicle remained the same. Therefore, a 2 step therapeutic approach might be an ideal treatment for CF. The first step would be composed of a compound that mimics the mechanism of stabilization provided by low temperature or the I539T mutation, while the second step would be VX-809 or a similar corrector compound. These studies suggest that understanding how low temperature and second site suppressors alter ΔF508-CFTR could be key to the development of future therapeutics for the effective treatment of CF. The precise pathophysiology of cystic fibrosis is not well studied. The involvement of another transport protein, epithelial sodium channel (ENaC), makes the situation more complicated. ENaC and CFTR are colocalized on the apical surface of epithelia cells. With our fluorescence microscopy techniques, we explored the effects of CFTR on the residence time of ENaC on the cell membrane. A reliable approach measuring the half-life of protein on the cell membrane is required for this study. We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Photoconversion of Dendra2 works as a pulse chase experiment by monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. The half-life of the protein on the plasma membrane was calculated upon imaging over time and quantifying the change in red fluorescence. Our method provides a unique opportunity to observe real-time protein turnover at the single cell level without addition of protein synthesis inhibitors. This technique will be valuable for the future protein half-life study